The present invention relates to high throughput screening (HTS) methods, e.g., for detecting the effect of a given compound or treatment on cell metabolic activity, and apparatus for performing such screening.
With the advent of combinatorial library methods for generating large libraries of compounds, there has been a growing interest in high-throughput screening (HTS) methods for screening such libraries.
The most widely used HTS screening method involves competitive or non-competitive binding of library compounds to a selected target protein, such as an antibody or receptor. Thus, for example, to select a library compound capable of blocking the binding of a selected agonist to a receptor protein, the screening method could assay for the ability of library compounds to displace radio-labeled agonist from the target protein.
Although such binding assays can be used to rapidly screen large numbers of compounds for a selected binding activity, the assay itself may have limited relevance to the actual biological activity of the compound in vivo, e.g., its ability to interact with and affect the metabolic behavior of a target cell.
It would therefore be useful to provide high throughput screening methods capable of testing the effects of large numbers of library compounds on target cells of interest.
The invention includes, in one aspect, high throughput screening apparatus, e.g., for screening the effect of test compound on cell metabolic activity, or for screening the effect of a genetic manipulations on cells. The apparatus includes a multiwell device defining a plurality of discrete microwells on a substrate surface, at a well density of greater than about 100/cm2, where the well volumes are such as to accommodate at most about 106 cells/well, preferably between 1-100 wells/cell, and structure for measuring the conductance in each well. The measuring structure includes (i) a pair of electrodes adapted for insertion into a well on the substrate, and (ii) circuitry for applying a low-voltage, AC signal across the electrodes, when the electrodes are submerged in the medium, and for synchronously measuring the current across the electrodes, to monitor the level of growth or metabolic activity of cells contained in the chamber.
In various preferred embodiments, the signal circuitry is effective to generate a signal whose peak-to-peak voltage is between 5 and 10 mV, and includes feedback means for adjusting the signal voltage level to a selected peak-to-peak voltage between 5 and 10 mV.
In other embodiments, the circuitry is designed to sample the voltage of the applied signal at a selected phase angle of the signal, or alternatively, to sample the voltage of the applied signal at a frequency which is at least an order of magnitude greater than that of the signal.
In another general aspect, the invention includes a high-throughput screening method, e.g., for screening the effect of test compounds on cell metabolic activity, or the effect of a given genetic manipulation. The method includes placing cells in the wells of a multiwell device defining a plurality of discrete microwells on a substrate surface, at a well density of greater than about 100/cm2, with the number of cells in each well being less than about 106, and preferably between 1-103. The conductance in each well is determined by applying a low-voltage, AC signal across a pair of electrodes placed in the well, and synchronously measuring the conductance across the electrodes.